Amino acids currently have application as additives to animal feed, nutritional supplements for human food, components in infusion solutions, and synthetic intermediates for the manufacture of pharmaceuticals and agricultural chemicals. L-glutamic acid is used as a flavor enhancer for food with a world market of over 1 billion dollars annually. L-lysine and methionine are large volume additives to animal feed, and L-tryptophan and L-threonine have similar potential applications. L-phenylalanine and L-aspartic acid have very important markets as key components in the manufacture of the low-calorie sweetener aspartame, and other promising low-calorie sweeteners have compositions containing certain amino acids as well. Infusion solutions require a range of amino acids including those essential in human diets. D-valine is used in the manufacture of synthetic pyrethroids. D-phenylglycine and derivatives thereof are useful as components of beta-lactam antibiotics. Many other D- and L-amino acids have potential applications as intermediates in the synthesis of pharmaceutically active compounds.
Methods developed for the synthesis of amino acids involve fermentation, chemical synthesis, extraction from protein hydrolyzates, and enzymatic bioconversions. Chemical synthetic methods generally involve the initial formation of a racemic mixture, followed by the resolution of this mixture to yield the optically active product. The resolution may be accomplished either chemically, by fractional crystallization of diasteromeric salts of the amino acid, or if desired, enzymatically using the enzyme L-aminoacylase. In order to make use of the undesired isomer, it must be re-racemized and then recycled through the process. Fermentation methods suffer from problems of slow rates of conversion, costly purifications, and significant capital investments. Extraction from protein hydrolyzates is used only in a few cases because the amino acid of interest is a relatively low percentage of the total protein and purification is therefore generally difficult. Enzymatic conversions offer advantages primarily due to reduced capital investments, lower purification costs, higher rates of conversion, and higher yields.
Although a number of biocatalytic routes have been proposed for the production of L-amino acids, few have been shown to be generally useful for the production of a wide range of different optically active amino acid products. For example, Chibata and co-workers at Tanabe Seiyaku as well as several other companies have developed a process for the production of L-aspartic acid from ammonium fumarate catalyzed by the enzyme aspartase. See Tosa, T. et al., Biotech and Bioeng. 15:69-84 (1973); Wood, L. L. and Carlton, G. J., Bio/technology 2:1081-1084 (1984); and Fusee, M. C. et al., Appl. Environ. Microbiol. 42:672-676 (1981). A process for the production of L-phenylalanine by the phenylalanine ammonia lyase-catalyzed addition of ammonia to trans-cinnamic acid has been developed. See Hamilton, B. K. et al., Trends in Biotechnology 3:64-68 (1985). L-Alanine can be produced by the enzyme-catalyzed decarboxylation of L-aspartic acid (see Fusee, M. C. and Weber, J. E., 1984, Applied and Environmental Microbiology 48:694-698, and references cited therein. However, none of the processes above has been shown to have truly general applicability to the production of a number of different amino acids, both naturally occurring and unnatural.
One previously described enzymatic process which does have general applicability involves the transamination of a given 2-ketoacid to the corresponding L-amino acid (U.S. Pat. No. 4,518,692 (May 1985)). In that process, L-aspartic acid and a 2-ketoacid are reacted in the presence of a transaminase to form the desired L-amino acid and oxaloacetate, followed by decarboxylation of said oxaloacetate to form pyruvate. The essentially irreversible decarboxylation of oxaloacetate drives the entire process to completion to form L-amino acids in yields approaching 100% of theoretical from the corresponding 2-ketoacids. The reaction is summarized in Scheme 1: ##STR1## The present invention is an improvement on the processes described in U.S. Pat. Nos. 4,518,692 and 826,766. The present invention provides a method for achieving the advantages of the transamination reaction, but allows the L-aspartic acid to be used in catalytic rather than stoichiometric quantities. L-Aspartic acid is thus replaced as the amino group donor by L-lactic acid, which is produced inexpensively in large quantity by fermentation. By replacing the L-aspartic acid with a less expensive precursor, L-lactic acid, the cost of raw materials for amino acid production is significantly reduced. The present invention affords additional advantages in that the reaction is driven to completion by removal of the oxaloacetate by reduction to L-malic acid rather than by decarboxylation. This eliminates the problems of evolution of gaseous CO.sub.2 and the concomitant pH changes associated with the production of CO.sub.2 as a byproduct. The present invention is also an improvement over US 4,304,858, DE 3307094, and DE 3307095 which disclose processes involving the reductive amination of 2-ketoacids to form L-amino acids. In the process of the present invention the net reaction catalyzed by the biocatalytic system is a reductive amination of a 2-keto acid with inexpensive L-lactic acid and ammonia to the corresponding optically active amino acid. One advantage of the present invention is that unlike the aforementioned inventions, no nicotinamide cofactors, either unmodified or modified, need be added to the reaction mixture.